JP2883085B2 - Glycoproteins of HCMV, antibodies thereto and methods of producing HCMV vaccines, and recombinant vectors therefor - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to human cytomegalovirus (HCMV).
And the production of the viral glycoproteins,
Vaccine potential and production of HCMV-specific antibodies
You. BACKGROUND OF THE INVENTION HCMV is a fairly important human pathogen and it
Need an effective vaccine against Conventional experimental
Vaccines are based on attenuated non-pathogenic forms of the virus
However, there are unwanted side effects. Book
The invention provides vaccines against HCMV using recombinant DNA techniques.
Provides another approach to the production of chin. Like other herpesviruses, HCMV is a multiple glycotan
Identify the protein (1,2). These characterizations are
CMV using lonal serum and monoclonal antibodies
Study of infected cells and purified virions
Including (2-10). One glycoprotein is partially purified
And has been shown to induce a neutralization response in guinea pigs.
I came. However, sugar sugars specified by HCMV
The total number of proteins is unclear and individual sugar
The vaccine potential of the protein is not known. By HCMV
Purification of individual glycoproteins from infected cells
It is not promising. Because the virus
Grows slowly, and during infection the host protein
Is not stopped. SUMMARY OF THE INVENTION The present invention is referred to herein as gB and gH.
Identification of DAN of HCMV encoding two glycoproteins and
Based on expression. gB protein is 137 from the F / D border
Hind III F of the HCMV genome, located between 8 and 4095 bases
Encoded by the DNA in the fragment. gH
Protein is located between 228 and 2456 bases from the L / D border
Encoded by the DNA in the Hind III L fragment
It is. According to one aspect of the present invention, a human HCMV neutralizing antibody
Or more antigen determinations that can enhance
The polypeptide incorporating the group is converted from a recombinant DNA vector.
A method comprising expressing in a suitable host product is provided.
Where the determinant (or determinants) is an F / D boundary
Between 1378 and 4095 bases from the HCMV genome
Encoded by DNA in Hind III F fragment
Corresponding to the protein part and / or from the L / D boundary
Between 228 and 2456 bases. Hind II of HCMV genome
Protein encoded by DNA in IL fragment
Corresponds to the quality part. A second aspect of the present invention relates to such polypeptides.
Provide recombinant virus vector containing DNA to be loaded
The vector can infect humans.
And expresses the polypeptide in an immunogenic form. A third aspect of the invention is to combine such polypeptides.
Providing a method comprising: A fourth aspect of the invention relates to such a polypeptide or
Host animal by the recombinant virus vector as described above
(Except for humans), and
Extraction of specific antisera from the host animal
For the preparation of HCMV monospecific antiserum comprising
You. HCMV-specific monoclonal antibodies are not
It can be prepared from cells from an infected animal. A fifth aspect of the present invention relates to an antibody and an HCMV polypeptide.
Contact and separate the bound antibody from the polypeptide.
Purifying an HCMV-specific antibody comprising releasing
Provide a way. A sixth aspect of the present invention relates to a sample and an HCMV polypeptide.
And binds to the polypeptide, and binds to the polypeptide.
HCMV in a clinical sample, comprising detecting the body
Methods are provided for detecting specific antibodies. A seventh aspect of the present invention provides such a detection method.
To provide a kit for
Is a suitable form of the polymer for contact with clinical samples.
HCMV-specific antibodies that bind to peptides and the polypeptides
Means for detecting the body. Identify HCMV surface glycoproteins that guide immune response
And the inheritance of its corresponding sequence in mammalian vectors
By introducing substances, vaccines against HCMV
An immunologically active tan that can form the basis
It can produce protein. As for the recombinant virus vaccine, its identification
HCMV genomic fragments are isolated and
Introduced into the appropriate mammalian virus vector by child engineering techniques
And transfer the plasmid into a mammalian host.
You can sfect. Suitable vectors are mammalian cells and viruses, e.g.
If poxvirus (particularly preferably vaccinia will
And bovine papilloma virus. Expression of exogenous DNA is achieved by a recombinant viral vector.
Obtained by infecting cells or animals. for example
For example, a recombinant virus, such as a vaccinia virus,
It can be used as a live vaccine. In addition, recombinant vectors
Used as a vaccine with cells infected by
The product of exogenous DNA can be prepared to
You. In one preferred technique, the glycoproteins of the HCMV genome
The coding fragment is introduced into the plasmid pG62.
And then infected by vaccinia virus
Transfect the plasmid into isolated mammalian cells.
Transfer during vaccinia virus
Is performed. HCMV DNA is
Can be modified in various ways without significantly affecting function
It will be clear that this can be done. For example, protein
The quality transmembrane form is the C
-Secreted form by removing the DNA encoding the ends
Can be converted to Such modifications are not claimed in the present invention.
Exists in the enclosure. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the proper cleavage for the restriction enzyme Hind III.
FIG. 3 is a map of HCMV genome strain AD169 in the prototype orientation,
Location and orientation of genes encoding CMV gB and HCMV gH
Also shown. FIG. 2 shows the HCMV gB gene identified in FIG.
Putative translation products and simple hell
Pesvirus type 1 glycoprotein B (as HSV
Named) and possible Epstein-Berau
Of the viral glycoprotein of the virus (named EBV)
Comparison with putative translation products. Possible glyco
The silation site is underlined and a putative hydrophobic sig
The null area and the anchor area are surrounded by solid lines. FIG. 3 shows the predicted amino acid sequence of HCMV gB, H
Hind I of the MCMV genome, including the gene encoding CMV gB
The HCMV genome, including the gene encoding the IIF flag gB
Xma III restriction enzyme fragment of Hind III F fragment
The DNA sequence of Clearly, this is
Shown in the opposite direction to the direction that exists in the prototype direction
You. FIG. 4 shows the introduction of the sequence of FIG. 3 into the plasmid pGS62.
Vaccinia in the future
It can be transferred into a virus. The dark line is HCMV
And the thin line indicates plasmid DNA. Open
Is the release box a vaccinia Hind III J fragment?
Axinia DNA taken from
Vaccinia promoter P in which
It has been translocated. FIG. 5 shows Sma extending the coding sequence for HCMV gH.
1 shows the DNA sequence of the I-Hind III L fragment. Fig. 3
This is in the direction of the prototype of the HCMV genome, as
In the opposite direction. HCMV gH
Putative amino acid sequence in single letter amino acid mode
Shown above the DNA sequence. S used for cloning
Restrictions for ma I (CCCGGG) and Hind III (AAGCTT)
The enzyme recognition sequence is underlined and its potential group
Lycosylation sites are also underlined. Hind III site
Is the boundary between the Hind III fragments L and D in the genome.
Show the world. Putative hydrophobic signal region and anchor region
The area is surrounded by a solid line. The present invention will be further illustrated by the following examples.
U. Example Identifying putative glycoprotein genes Identify possible glycoprotein genes in the HCMV genome
Due to the individual clones of the genome of the HCMV strain, AD169
Restricted fragment was identified in reference (11) as M13 /
Deoxynucleotide chain termination method or Ba in reference (12)
Uses the strategies and methods described by nkier and Barrell
And sequenced. Then the resulting sequence can be
Protein coding sequence and RNA polymerase II translocation
The transcript signal was analyzed. Next potential protein
Putative translation products of the quality coding sequence
Characteristic, that is, an N-terminal hydrophobic signal peptide,
Hydrophobic transmembrane sequence near the C-terminus and external
Existence of potential N-glycosylation sites in domains
Was tested. Using these criteria, the HindIII F flag of the HCMV genome
Between 16255 and 18972 bases of the HCMV genome
Present between 228 and 2456 bases of III L fragment
Two putative glycoprotein genes were identified. HC
The Hind III fragment map of the MV genome is shown in FIG.
Have been. In FIG. 1, each vertical dotted line is a long line.
And short unique regions. Uppercase letters in Hind III
Refers to the fragment produced by cleavage by
See reference (13). Glycoprotein gene B and
The position and orientation of H
Are shown in FIGS. Glycoprotein B code
The region extends from the Hind III F / D border on the complementary strand of the DNA sequence
Between bases 1378 and 4095 of glycoprotein H;
The coding region is on the Hind III L / D border on the complementary strand of the DNA sequence.
Located between bases 228-2456 from the kingdom. Glycoprotein B of HCMV Glycoprotein B gene in the indicated reading frame
Of the primary translation product of 16 potential N-linked
906 amino acid polypeptide containing a lycosylation site
It is. Near the N-terminus, acting as a signal sequence
A C-terminal that functions as a hydrophobic sequence and an anchor sequence
There are extensions of hydrophobic amino acids at the ends. Of this gene
Putative translation products are converted to other human herpesviruses.
Compared to glycoprotein gene. This survey is a simple
Pesvirus (HSV) and Epstein-Barr virus
(EBV) is homologous to glycoprotein B (gB)
Eagle (references 14); varicella and herpes zoster (VZ)
V) also has a homologous glycoprotein gene. this
For reasons, the tamper coded by this reading frame
The protein is substantially referred to as HCMV gB. Its putative translation products, HCMV gB and HSV1 and EBV
A comparison with them is shown in FIG. Estimated HC
MV glycoprotein sequences are those found in EBV and HSV1
Is consistent with the ones. These arrays are single-character
Optimal alignment of homologous amino acids as indicated by the amino acid code
Aligned and dashed to produce the best possible match
Paired by Regions with little homology, for example
At the end, the alignment is arbitrary. Characteristics of glycoprotein
The hydrophobic a at the N-terminus and near the C-terminus
The amino acid region is boxed with a solid line, and a possible N-
Compatible glycosylation sequence (N ^* T or N ^* S; * which one?
Are underlined. FIG. 2 shows good gB proteins of HSV-1, EBV and HCMV.
Show consistency and therefore their protein is minimally preserved
With their N- and C-termini,
Indicates that the parts are the same. All three proteins
In quality, at position 121, an equally matched amino acid is
It can be seen that it exists. Take as gB part of EBV
If this is the case, this is preferably at least 14% of the protein
Means saved. In addition, putative signal
All 10 existing between the
The cysteine residues are preferably aligned and
The extracellular portion of the protein has all similar structures.
Suggest that you can. These three kinds of windows
The degree of homology between the lus proteins depends on the putative HCMV sugar.
Belief that protein is a homolog of glycoprotein B
Provide proof of evidence. The characteristics of that glycoprotein
One evidence is that its characteristic hydrophobicity is shown in FIG.
Region and potential N-glycosylation sites
Provided. To study the gB properties of HCMV and this protein
Hind I in the HCMV genome to raise antiserum to quality
Cut out the gene from the II F fragment and recombine
It was expressed in vaccinia virus. This vector system
For the expression of eukaryotic viral glycoprotein genes
Is appropriate. Because the protein is
And inserted into the infected cell membrane
Because. In addition, infectious recombinant viruses are
To foreign proteins in inoculated animals
Can be used to raise monospecific antisera. The coding sequence shown in FIG.
Introduced into the Xenia virus. Shown in FIGS. 2 and 3
Sequence is commonly used in the coding sense sequence
Shown in 5 'to 3' form. This is shown in FIG.
In the opposite direction to the direction of the HCMV genome prototype. HC
GB amino acid sequence of MV uses single letter amino acid code
And is shown above the DNA sequence. Construction of HCMV gB expressing recombinant vaccinia virus 1. Strategies Expression of exogenous gene in vaccinia virus
Depends on the use of vaccinia promoters (see
See ref. 15). This is a vaccinia promotion
Unique properties of RNA polymerase and RNA polymerase II
Vaccinia DNA vectors that do not recognize the transcribed promoter
Because of the presence of limalase. Vaccinia virus
Designed to promote exogenous gene expression in
Some plasmids have been constructed (see
See references 16-18). They are vaccinia professionals
Transcription within the motor and thymidine kinase (TK) genes
Slowed downstream restriction endonuclease site
including. Next, the coding sequence for the foreign protein is
Located downstream of the senior promoter, and
Insert into vaccinia genome by homologous recombination method in vivo
(See reference 17). Vaccinia pro
Motor coding sequence and foreign protein coding sequence
Is the translation initiation codon of the foreign gene
If manufactured to use
Parkin is produced. Nucleotide sequence of the HCMV gB gene and adjacent DNA.
Test for the presence of the restriction endonuclease Xma III site
And 148 nucleosides upstream of its gB coding sequence.
And 251 nucleotides downstream of it. Further
Next, the upstream Xma III site and the ATG
No possible translation initiation codon between
Was. Therefore, this strategy requires that the 3.1 Kb fragment
The gB gene is cut out from the CMV Hind III F fragment,
This fragment is then inserted into the plasmid insertion vector pGS6.
2 (derivative of pG620-see ref. 17-here
EcoRI site upstream of the Xinia promoter has been deleted.
) At the Sma I site. this
In the method, the gB gene replicates vaccinia virus
Of the vaccinia promoter expressed throughout the cycle
Will be under control. Direct fragment of the desired fragment
The unit is the large size of the HCMV Hind III F fragment.
And difficulties due to the presence of other Xma III sites. 2. Experiment More specifically, the coding sequence of Fig. 3 is an example in Fig. 4.
Vaccinia Will by the following series of operations shown
Was introduced during the a) In the truncated pAT153 (see reference 19)
The cloned Hind III F fragment was transferred to BamHI.
Digestion, and separate the products by electrophoresis.
After isolation of the 8.5 Kb fragment and self-ligation
At least, plasmid pSB1 (also known as pSCB1)
). pSB1 is the 3.5Kb Hind III / BamHI of pAT153.
5Kb Hind III / BamH cloned into fragments
Includes I fragment. b) digest pSB1 with BamHI and HindIII and 5.
The HCb fragment of 0 Kb was isolated and
Digested. The resulting 3.1 kb Xma III fragment
The 5 ′ overhang was isolated and the DNA polymerase I
Repaired by the Know fragment and the repaired
The fragment was ligated into the SmaI site of plasmid pGS62.
Was. pGS62 is driven by the vaccinia promoter element.
Vaccinia virus thymidine kinase (T
K) Including genes. Insertion of a foreign gene at the Sma I site
Results in a plasmid where its exogenous HCMV gB
The gene is under the control of the vaccinia promoter (P).
And end with the thymine dikinase coding sequence
You. The orientation of the gB gene in the resulting plasmid pSB2 is
Unique from 3 'Xma III site as a convenient marker
Determined using 911 nucleotides with a unique EcoR I site
Was. Recombinant plasmids are identified by increasing size,
And the orientation of the 3.1K insert is determined by digestion with EcoR I.
It has been determined. Right about vaccinia lomotor
Identify plasmids containing an Xma III fragment in the orientation
And call pSB2 (also known as pSCB2)
Named. CV-1 cells were isolated from the cells using the method described in Reference 17.
Infected by Xinia virus, and by pSB2
Transfected. The recombinant virus is
TK for insertion inactivation of the near TK gene ^- And then
The leverage phenotype provides a means for easy isolation
Was. TK ^- The virus contains 5-bromodeoxyuridine.
143-TK in the presence ^- By plating on the cells,
Selected from the resulting offspring of, and such will
The sclone is hybridized with pSB1
Screened for the presence of the HCMV-specific DNA insert
Was. After propagation and production of the virus, the recombinant virus
Nome is digested with restriction endonuclease and Southern
Analyzed by the method. The result shows that HCMV gB gene is Hin
Insertion into vaccinia TK gene in dIII J fragment
And that other genomic rearrangements have occurred.
It was not shown. HCMV gB
-VAC. Expression of HCMV gB by recombinant vaccinia virus To test for expression of HCMV gB, purified HCMV was
HCMV using polyvalent rabbit serum
From cells infected by gB-VAC or WT vaccinia
of, ³⁵ Immunoprecipitation of S-labeled polypeptide
Squeezed. CV-1 cells were transformed with WT vaccinia or recombinant HCMV gB-VAC.
30 plaque forming units per cell (pf
Infected by u). 3 hours after infection, the cells
Was washed with methionine-free medium, and
Incubate in methionine-free medium for 30 minutes.
I did it. Transfer the cells to 10 μM unlabeled methyl
100 μCi / ml in a medium containing onin ³⁵ S-methionine
Labeling. After washing with PBS, the cells are
RIPA buffer (0.05M Tris-Hcl pH 7.2, 10 min on ice)
0.15M NaCl, 1% sodium deoxycholate, 0.1%
SDS, 1% Tritonx-100, 5 μg / ml DNase, 2 mM PMS
Decomposed by F). The decomposed product was heated at 4 ° C for 60 minutes at 31,00
Centrifuge at 0 rpm (Beckman SW50.1 rotor) and
An aliquot of the resulting supernatant is highly immunized with HCMV.
Serum from immunized rabbits or non-immune rabbit serum
Incubated together (20 minutes at room temperature). Immune complex
Precipitate the body with Protein A Sepharose (2 o'clock)
Elute, boil, and add 10% polyacrylic acid
The gel was electrophoresed on a luamide gel. The gel is methano
In acetic acid / acetic acid, and
Impregnate with Hanser (Amplify, Amersham)
To prepare an autoradiograph. Infected by HSV-1
Molecular weight markers for proteins from isolated cells
Capsid antigen (157KD), glycoprotein T (128KD)
And monoclonal antibody to VP16 (66KD)
I was confused. Using immune serum, a polypeptide of about 145 KD was
Cells infected with gB-VAC (sensitized by WT vaccinia)
(Not stained cells). Other ports
Re-peptides, their reactivity with pre-immune rabbit sera
Nonspecifically precipitated as indicated by
Was. Next, it was born against HCMV and
(B) A murine monochrome that has been shown to have neutralizing activity
HCV in cells infected with HCMV gB-VAC.
Test their ability to recognize MV gB products
Tested. HCMV gB-VAC on the monolayer of CV-1 cells
The formed plaque is fixed with methanol and
And then the monoclonal antibody and then ¹²⁵ From IN
Labeled Staphylococcus
Protein A (Amersham) or peroxidase conjugation
Rabbit anti-mouse immunoglobulin (DaKo)
Incubated together. By monoclonal antibody
Virus plaques containing the recognized antigen
In the case of A-binding antibody, the black dot on the autoradiograph
React with peroxidase-conjugated antiglobulin
H to single layer _Two O _Two And addition of amino-ethylcarbazole
And visualized as "red plaque" according to Tested
Four of the 10 monoclonal antibodies were HCMV gB
-A plug formed by VAC (not WT vaccinia)
To recognize the work. Items indicating HCMV gB-VAC
Clonal antibody conjugated, formed by the virus
All of the plaques that were removed are pure and WT wax
It should be noted that it is not contaminated by near.
Similar conclusions are drawn to the presence and absence of bromodeoxyuridine.
TK in the presence ^- Plaque by virus on 143 cells and then
Obtained by analysis of genomic DNA by Southern method. WT vaccinia, recombinant HCMV gB-VAC or infected
Cell lysates from free CV-1 cells were prepared and
As described, by monoclonal antibody 37, 39 or 59
It was immunoprecipitated. Three types of monochrome that could bind protein A
The internal antibody also ³⁵ Labeled with S-methionine
Infectious cell extracts were also immunoprecipitated. Monochrome
Null antibodies 37 and 39 were HCMV gB-VAC (but WT vaccinia)
Or not uninfected cells)
Immunoprecipitation of proteins from cells means that
Consistent with staining data. Monoclonal antibody 59, HCMV
Although it can neutralize infectivity, this antibody does not
MV gB was not recognized. The target protein for this antibody is
unknown. Approximately 55KD in addition to 145KD protein
Lower molecular weight proteins are also
Detected by the body. This is both a monoclonal antibody
Epitopes recognized by both are 55KD and 145KD
Are present on the protein, or these proteins are physically
Relevant and consequently suggest co-precipitation. GB and HCMV g synthesized in cells infected by HCMV
Direct comparison with gB from cells infected by B-VAC
To achieve this, MRC-5 cells were transformed with HCMV strain, AD16 at 5 pfu / cell.
Infected by 9 or pseudo-infected. 72-98 hours
Later, cells from the infection, ³⁵ S-methionine (28μCi / ml)
And prepare a degradant, and
As in either monoclonal antibody 39 or 47
More immunoprecipitation. WT vaccinia, recombinant gB-VAC
CV-1 cells infected or uninfected by
Radiolabel, disassemble, and
Immunoprecipitation was performed with clonal antibody 39. 145 KD species synthesized in both systems are clearly simultaneous
The migrated and mature 55KD species also have similar sizes
Non-specific precipitation of vaccinia band in the bacterium makes this clearer
I did make sure, but they moved clearly at the same time. These two types
In addition to the types, 66KD band is also more sensitive to HCMV
It was also found in stained cell lysates. This is unrelated to gB
Seems to be in charge. Because it binds protein A
Another monoclonal antibody (47) that did not
This is because the band was specifically caused. That rhino
Is a lot of 66KD HCMV matrix tamper
Suggested that Human immune serum recognizes HCMV gB. Antibodies directed against HCMV gB have been identified in humans
To determine whether it is produced during the next HCMV infection,
Patients who have heart transplants before and after primary HCMV infection
Serum collected from recombinant vaccinia virus
Tested for its ability to recognize synthesized HCMV gB
Was. Infected by either H3-VAC or HCMV gB-VAC
CV-1 cells were prepared as described above, ₃₅ S−
Labeled with methionine cell lysate. Next, the decomposition product
With rabbit play immunization followed by rabbit anti-CHMV or HCMV
Human serum collected prior to infection with
Were continuously treated with any of the sera after infection.
Immune complex is precipitated by protein A sepharose
And polyacrylamide, as described above.
Separated on the Rabbit sera raised against HCMV were
Used as illumination. Human serum was also
Contains antibodies to vaccinia virus for chin
Because of this tendency, immunoprecipitation is
Followed by the same cell lysate using serum collected later.
Was performed continuously. These data are 145KD
In some cases, the polypeptide may be present after HCMV infection (but not before).
G) Infected by gB-VAC with collected human serum
Shows that immunoprecipitation was performed from the
You. The 145 KD protein is also available in rabbit anti-HCMV serum.
Therefore, it was precipitated. As a further control,
Serum is another recombinant vaccinia virus H3-VA
Influenza virus A / NT / 60/6 expressed by C
Influenza virus hemagglutinin (HA) from 8
Tested for ability to recognize. The flu
HA is immunized with human serum collected before HCMV infection
Serum precipitated and collected after HCMV infection
Not so much. These data are
H3 subtype influenza received in heart transplant patients
A indicates previous experience of A virus infection. Furthermore, HC
MV gB was simply precipitated by serum collected after HCMV infection.
Blood collected before HCMV infection
Precipitation of influenza hyaluronan by Qing
Confirm the specificity of the precipitate. The growth of antibodies against HCMV
Does immunosuppression during heart transplantation prevent tissue rejection?
Nevertheless, what happened is also significant. Human immune serum
It can also neutralize HCMV infectivity in vitro.
Wear. Heart transplant, who experienced HCMV infection during immunosuppression
Some other sera collected from the recipient patients also
A similar precipitation of HCMV gB resulted. HCMV gB is expressed on infected cell surfaces In cells infected with recombinant vaccinia virus
The HCMV gB synthesized in the cell is transported to the cell surface
An immunofluorescence study was performed to test for suspicion. CV-1 cells were grown on glass coverslips
WT vaccinia virus or HCMV at 10 pfu / cell
Infected by any of -gB-VAC. 48 hours after infection
Between the cells with an isotonic solution of 2% paraformaldehyde.
Fixed. PBS containing 4% bovine serum albumin (BSA)
After incubation in the monolayer at 4 ° C. overnight,
1/400 dilution of monoclonal antibody 37 containing ascites
Reacted. After extensive washing 4% bound antibody
1/20 dilution with PBS containing BSA and 2% normal rabbit serum
Fluorescein-conjugated rabbit anti-mouse
Detected by Roblin (DaKo). Fluorescent, x400 uv
Observed by irradiation. Cells infected with HCMV gB-VAC have positive surface fluorescence.
Cells that showed light, but were infected by WT, were eyes
No reactivity was shown. Staining on infected cell membranes
The pattern usually shows a granular appearance and
HCMV gB aggregation or aggregation. Vaccination of rabbits with HCMV gB-VAC HCMV g expressed by recombinant vaccinia virus
Anti-serum raised against B neutralizes HCMV infectivity
Two animals to determine if they can
The heron was treated with live recombinant virus as shown in Table 1.
Vaccination. Two rabbits in one site on each flank
In addition, purified infectious HCMV gB-VAC 10 ⁸ subcutaneous by pfu
Immunized. 46 days later, both animals are treated with the same dose of recombinant
Re-vaccinated with the vaccine. The third rabbit,
Expresses the influenza virus nucleoprotein gene
TK ^- Receiving recombinant vaccinia virus
Vaccinated. On the day shown in Table 1,
The serum sample obtained is used for in vitro HCMV infectivity.
Were tested for their ability to neutralize. Serum sump
Ink at 56 ° C for 30 minutes to inactivate complement.
And then a serum dilution (1:10 or 1: 5
0) and an equal volume of HCMV strain AD169 (750pfu)
And incubated at 37 ° C. for 30 minutes. New c
Egg serum was added as a source of complement to a final concentration of 5%,
And incubate the mixture at 30 ° C for another 30 minutes
And then determine residual virus against MRC-5 cells
did. Plaques were counted after 10 days and the results
Are shown in Table 1 as% reduction in plaque count.
Serum from rabbits 1 and 2 was purified from HC1 in the presence of exogenous complement.
Antibodies that neutralized the infectivity of MV were included. Different TK ^- Recombination
Third incubated with vaccinia virus
Rabbits did not have such antibodies. Therefore, two animals vaccinated with HCMV gB-VAC
Animals grow antibodies that neutralize HCMV in vitro.
But the protein of influenza virus
Another TK to be expressed ^- By recombinant vaccinia virus
The third immunized rabbit was not. these
Neutralization of HCMV infectivity by rabbit serum is present in complement.
Was. Because serum that has been inactivated by heat, exogenous
Without the addition of sex complement, the number of HCMV plaques was not reduced.
Test antibody levels before and after the second vaccination.
Another experiment to test was that both animals were revaccinated.
Later, it was shown that the antibody titer was increased. Rabbit 1 is 116
Maintain that antibody level until day and at this point,
A 1:50 dilution reduced HCMV plaque formation by 70%. Rabbit
The antibody level in 2 decreased with time, but only
Day 116, 24% HCMV plaque formation at a dilution of 1:50,
It has been reduced. Conclusion The putative coding sequence is the true expression of the vaccinia
Encodes HCMV protein. The expression of vaccinia
Protein is a protein found in cells infected by HCMV.
It was found to be identical by protein and electrophoresis. So
Has the following properties: It neutralizes antibodies
(Because it is a neutralizing
Because it is recognized by the body). It is HCMV gB
-On the surface of cells infected by VAC, and
It is present in HCMV particles. The primary product has an apparent molecular weight of 145,000 and 5
Processed to 5,000 molecular weight product. It is a recombinant
Through expression in cells infected by the virus
Antibodies that neutralize HCMV infection when released to the rabbit immune system
Can be induced. The above example used HCMV strain AD169, but other bacteria
Strains are also functionally equivalent and can also be used
You. Glycoprotein H of HCMV Putative amino acid of the gene as shown in FIG.
Comparison of acid sequence with amino acid sequences of EBV and HSV-1 genes
Indicates that the glycoproteins H of these viruses are homologous.
Was shown. Therefore, this HCMV gene is referred to as HCMV gH.
Is referred to. Construction of recombinant vaccinia virus expressing HCMV gH The HCMV gH gene was converted into a plasmid vector as follows.
-Cloned into pGS62. 11400 base pairs Hind I
The II L fragment was purified by digestion with Hind III.
Cut from Smid pAT153 / Hind III L and E. coli
Of DNA with DNA polymerase Klenow fragment
Therefore, their ends were blunt-ended. Next, the DNA
As shown in FIG. 5, the translation disclosure section of the CMV gH gene.
By Sma I, which cuts 96 nucleotides upstream of the position
Digested. 2.5Kb DNA flag containing HCMV gH coding sequence
Units and plasm at unique Sma I sites
Ligated into pGS62. The resulting plasmid pSB3 is
Correctly positioned downstream of the Xinia promoter
It was shown to contain the HCMV gH gene. The method comprises H
Essentially similar to the method used for the CMV gB gene
Was. The HCMV gH gene also has its same vaccinia promoter.
At the unique SAm I site downstream of the plasmid pSC11 (see
Reference 16). This plasmid is called pSB4
Was. Plasmid SC11 contains the β-galactosidase gene
And a second vaccinia promoter that directs the expression of
Therefore, a recombinant virus that simultaneously obtains the HCMV gH gene is
It also acquires the β-galactosidase gene. This
This is due to their blue color in the presence of X-gal
Of plaques formed by recombinant viruses
Enable identification. Using plasmids pSB3 and pSB4, the HCMV gH gene was
Including TK ^- A recombinant vaccinia virus was constructed. Those
Viruses are HCMV gH-VAC (GS62) and HCMV, respectively.
It was called gH-VAC (SC11). These viruses are
Lark refined and then more stock
Proliferated and grown using the established method (Ref. 17).
And purified. Analysis of the genomic DNA of those viruses
Indicates that the HCMV gH gene is vaccinia Hin
Inserted into the TK gene with the d III J fragment
That was shown. Expression of HCMV gH gene The product of the HCMV gH gene was purified as follows.
Identified by cells infected by VAC: (1) Monolayers of CV-1 cells were treated with WT vaccinia (WT) or
Replacement vaccinia virus CMV gH-VAC (GS62) or C
Infection was caused by any of MV gH-VAC (SC11). Feeling
Stained cells 3-6 hours after infection ³⁵ S-methionine
Radioactively labeled and degraded for 6 hours post-infection
Prepared from the infected cells. These decomposition products
To non-specific rabbit serum, purified HCMV virions
Rabbit serum expressed against or anti-HCMV monoclonal
Immunoprecipitation with either antibody 16 (HCMV 16)
Was. About 86KD polypeptide, using rabbit anti-HCMV serum
Infection by gH-VAC (SC11) and gH-VAC (GS62)
The cells were immunoprecipitated. This peptide is
Precipitated from cells infected by vaccinia
Did not. For synthetic peptides derived from HSV glycoprotein D
The expressed rabbit serum precipitates this band.
Did not. However, similarly sized polypeptides
Were infected with HCMV using rabbit anti-HCMV serum.
From the harvested MRC-5 cells. (2) Anti-HCMV monoclonal 16 is also gH-VAC (however,
86 KD band from cells infected with WT
To confirm HCMV gB immunoprecipitated
HCMV37 precipitated 86 KD protein as a control.
I didn't. (3) synthesized in cells infected by HCMV gH-VAC
Cell location of HCMV gH was determined by immunofluorescence.
Was. This means that the gH polypeptide is transported to the nuclear membrane, and
In addition, it was detectable diffusely in the cytoplasm
showed that. If cells are not initially permeated, HCMV g
No fluorescence on cells infected by H-VAC
Was. Monoclonal HCMV 16 neutralizes HCMV infectivity HCMV gH is used for antibody-mediated neutralization of viral infectivity
HCMV to determine if it is
Incubate with Lonal HCMV 16 and remain
Infectivity was assayed on MRC-5 cells. 1: 4000 dilution
Even to a degree, monoclonal HCMV 16 is H
CMV infectivity was reduced by more than 50%. This neutralization is caused by exogenous complement
Did not depend on. Clearly, the product of the HCMV gH gene
Is a target for virus neutralization and
Thus, there is potential for future HCMV vaccines. Conclusion Mapped within the HindIII L fragment of HCMV
The DNA sequence of the HCMV glycoprotein gene has been determined, and
Expressed in recombinant vaccinia virus. The gene student
The product is produced in cells infected by the recombinant vaccinia.
Transported to the nuclear envelope. Identified as 86KD polypeptide
Was. In cells infected by HCMV,
It is also present on cell surface membranes. Recognize this protein
Monoclonal antibodies can be used to infect HCMV in vitro.
To effectively neutralize. This is the HCMV vaccine
This shows a possible role for the 86KD glycoprotein. The above description indicates that the virus was
Of HCMV protein in isolated cells and protection against infection
Acts as a vaccine that causes the host to produce one of the antibodies
By way of example, against the possibility of the virus
As a result, the present invention is readily applicable to the species
It is clear that modifications can be made by a variety of different techniques.
It will be clear. These are exemplified as follows. (I) DNA and amino acid sequences given in FIGS. 3 and 5
Based on HCMV gB and gH proteins
In a manner well known in the art
Can be. For example, encode the desired amino acid sequence
DNA can be synthesized. On the other hand, the DNA is restricted,
Subsequent interaction with the labeled oligonucleotide probe
Identifying the sequence of interest by hybridization
From the viral genome. In addition, cDNA
Reverse transcription from lus mRNA, followed by oligonucleotide high
Screening with hybridization probe
Obtained by: (Ii) HCMV protein is formed by recombinant DAN vector
Expressed in transformed microorganisms or cell cultures. Appropriate
Vectors and expression systems are widely known and
For example bacteria, such as E. coli, yeast,
For example, Saccharomyces cerevisiae (Saccharo
myces cerevisiae) and mammalian cell cultures, such as CO
Used to express proteins in S or CHO cells
You. For microbial expression (eg, bacteria and yeast), HCMV
Before insertion into the expression vector,
Normal operation to remove the 5 'flanking region
The coding region is the ATG or HCMV protein gene
Is artificially introduced by matching its coding sequence with the reading frame
Translated from the start codon, one of the ATGs. Co
The 5 'region of the primer sequence, eg, the hydrophobic signal region
If it is desired to use the latter,
There will be. The hydrophobic 3 'anchor region is also optional.
Can be removed. Because it is a critical antigenic determinant
This is because there is a tendency not to include. The recombinant vector
Is the coding sequence for multiple HCMV proteins, e.g.
GB or gH coding sequence in dem repeats or gB in tandem
And gH coding sequences. Expression
In order to reduce the size of the
Only a part of the coding sequence of the protein
It can be introduced as long as the group is correctly coded. (Iii) HCMV protein or desired antigenic determinant
Some of which also use known methods of protein synthesis.
And can be synthesized by chemical means. (Iv) However, the produced HCMV protein
Immunized animals with HCMV protein and
Allows the production of antibodies against the protein, and then
Extract antiserum from the
Can be used to produce HCMV-specific antibodies.
You. (V) the HCMV protein is an animal derived from the protein,
Usually immunization of mice, followed by isolation and cloning
To form an antibody-producing hybridoma that can be
Standard techniques for fusion of tumor cells with spleen cells from the animal
Produce HCMV-specific monoclonal antibodies
Can be used especially for From these clones,
Clonal antibodies can be harvested. Usually, antibodies are
Produced. Because each HCMV protein
May be expected to produce multiple antibodies.
You. (Vi) HCMV proteins can also be used on suitable supports, such as
Protein and antibody immobilized on the affinity column
And then e.g. by elution
Separation of bound antibody from other proteins
Can also be used to purify HCMV-specific antibodies.
You. (Vii) HCMV protein also assay for HCMV antibody
Can also be used. Various conventional assay methods are available.
For example, it can be used based on ELISA, RIA or immunofluorescence.
You. Typically, HCMV proteins are immobilized on a support.
Can then be contacted with a clinical sample from a human. Washing
After purification, the support is converted to immobilized HCMV protein.
Thus, the label that binds to any HCMV antibody
Contacted anti-human IgG. (Viii) HCMV proteins can also be used in vaccine formulations
With a suitable adjuvant or excipient of the type normally used
By mixing them together, they can also be used as vaccines.
Can be used. The form of this vaccine is
Are more appropriate than recombinant vaccines in
There will be. References 1. Stinski, M. (1976) J. Virol. 19 2. Pereira, L., Hoffman.M., Tatsuno, M and Dondero, D.
(1984) Virology, 139 3. Pereira, L., Hoffman, M. and Cremer, N. (1982) Infe.
ct.Immun., 36 933-942. 4. Pereira, L., Hoffman, M., Gallo, G, and Cremer, N. (1
982) Infect.Immun., 36 , 924-932. 5. Britt, WJ (1984) Virology, 135 , 369-378. 6. Nowak, B., Sullivan, C., Sarnow, P., Thomas, R., Brico.
unt, F., Nicolas, JC, Fleckenstein, B.and Levine, AJ
(1984) Virology, 132 325-338. 7. Law, KM, Wilton-Smith, P. and Farrar, GH (198
5) J.Med.Virol. 17 Rasmussen, L., Mullenax, J., Nelson, R. and Merigan,
TC (1985) J. Virol. 55 Rasmussen, L., Mullenax, J., Nelson, M. and Merigan,
TC (1985) Virology, 145 10. Britt, WJand Auger, D. (1986) J. Virol., 186-190. 58 , 185
−191. 11. Sanger, F., Coulson, AR, Barrell, BG, Smith, A.
JHand Roe, BA (1980) J. Mol. Biol., 143 , 161-178. 12. Bankier, AT and Barrell, BG (1983). Shotgun D
NA sequencing in Techniques in the Life Sciences.v
olume B508, pp. 1-34.Elsevier Scientific Publishers
Ireland Ltd. 13. Oram, JD, Dowing, RG.Akrigg, A., Dollery, AA,
Duggleby, CJ, Wilkinson, GWGand Greenaway, PJ
(1982) J. Gen. Virol., 59 , 111-129. 14. Pellet, PE, Biggin, MD, Barrell, BGand Roizm
an, B. (1985) J. Virol. 56 15, 807-813. 15.Smith, GLand Moss, B. BioTechniques 2,120-12
6, (1984). 16. Charkrabarti, S., Breckling, K. and Moss, B. (198
5) Mol. Cell. Biol. 5 , 3403-3409. 17. Mackett, M., Smith, GLand Moss, B. (1984) J. Vir.
ol, 47 18.857-864. 18. Boyle, DB, Douper, BEHand Both, GW (1985)
Gene, 35 Twigg, AJand Sherratt, D. (1980) Nature, 283 , 2
16.

──────────────────────────────────────────────────続 き Continued on the front page (31) Priority claim number 8629988 (32) Priority date December 16, 1986 (33) Priority claim country United Kingdom (GB) (72) Inventor Barrel, Barclay George Stancegate Avenue, Cambridge, UK 16 (56) References JP-A-61-22100 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/00-15 / 90 EPAT (QUESTEL) BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] Encodes the following polypeptide a) or b)
DNA: a) HCMV glycoprotein gB or gH for human cytomegalovirus (HCMV) strain AD169, which has the amino acid sequence shown in Sequences 1 and 2 below: Or b) has an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence a), and has the antigenicity of HCMV glycoprotein gB or gH.
HCMV glycoprotein gB or gH. 2. DNA having the following DNA sequence of a) or b): a) DNA sequence shown in the following sequences 1 and 2 for human cytomegalovirus (HCMV) strain AD169; Or b) a DNA sequence that hybridizes under stringent conditions to the DNA sequence of a) and encodes a polypeptide having the antigenicity of HCMV glycoprotein gB or gH. 3. Deletes all or part of the C-terminal membrane anchor sequence
The DNA according to claim 1, which encodes HCMV glycoprotein gB or gH. 4. Deletes all or part of the C-terminal membrane anchor sequence
The DNA according to claim 2, which encodes HCMV glycoprotein gB or gH. 5. The DNA according to claim 1, wherein the HCMV glycoproteins gB and gH have the amino acid sequences shown in Sequences 1 and 2. 6. Having the DNA sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2,
The DNA according to claim 2. 7. Encodes the following polypeptide a) or b)
A recombinant vector comprising DNA, which is capable of expressing the polypeptide: a) HCMV having the amino acid sequence shown in the following sequence 1 and 2 for human cytomegalovirus (HCMV) strain AD169 Glycoprotein gB or gH; Or b) has an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence a), and has the antigenicity of HCMV glycoprotein gB or gH.
HCMV glycoprotein gB or gH. 8. A recombinant vector comprising a DNA having the following DNA sequence of a) or b), capable of expressing a polypeptide encoded by the DNA: a) Human cytomegalovirus (HCMV) strain AD169 DNA sequences shown below in SEQ ID NO: 1 and SEQ ID NO: 2; Or b) a DNA sequence that hybridizes under stringent conditions to the DNA sequence of a) and encodes a polypeptide having the antigenicity of HCMV glycoprotein gB or gH. 9. The recombinant vector according to claim 7, wherein the HCMV glycoproteins gB and gH have the amino acid sequences shown in Sequences 1 and 2. 10. D having the DNA sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2
9. The recombinant vector according to claim 8, comprising NA. 11. A recombinant vector capable of infecting a mammalian subject,
The recombinant vector according to claim 7 or 9. 12. A recombinant vector capable of infecting a mammalian subject,
The recombinant vector according to claim 8 or 10. 13. 12. The recombinant vector according to claim 11, wherein said mammalian subject is a human. 14． 13. The recombinant vector according to claim 12, wherein said mammalian subject is a human. 15. 14. The recombinant vector according to claim 13, suitable for use in a vaccine against HCMV. 16. 15. The recombinant vector according to claim 14, suitable for use in a vaccine against HCMV.